Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
  • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

• the present invention provides chemokines called human LI 05 proteins and polynucleotides encoding them.
• Chemokines are a subclass of cytokines which cause the directed migration or chemotaxis of particular cell populations either to or away from higher concentrations of the chemokine. Many chemokines have been identified which cause migration of major blood cell populations. These factors may be useful for directing the migration of cell populations to areas of desired action or away from areas of unwanted action. It would, therefore, be desirable to identify new chemokines and polynucleotides encoding them.
• WO97/07198 disclose a novel murine chemokine identified as "L105.”
• Murine L105 has been demonstrated to have chemokinetic/chemoattractant activity for several cell types, including thymocytes, kidney mesangial cells and lymphocytes localized in lymph nodes. Because of these beneficial activities, it would be desirable to identify human homologues of murine SUMMARY
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
• polynucleotide which is an allelic variant of SEQ ID N0:3; and (o) a polynucleotide capable of hybridizing to any one of the polynucleotides specified in (a), (b), (d), (e), (h), (i), (k) or (1) above.
• such polynucleotide comprises the nucieotide sequence of SEQ ID NO: 1 from nucieotide 80 to nucieotide 481; the nucieotide sequence of SEQ ID NO:l from nucieotide 119 to nucieotide 481; the nucieotide sequence of the full-length protein coding sequence of clone huL105_3 deposited under accession number ATCC 98429; or the nucieotide sequence of a mature protein coding sequence of clone huL105_3 deposited under accession number ATCC 98429.
• the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone huL105_3 deposited under accession number ATCC 98429.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having chemokine activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:2, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having chemokine activity, the fragment comprising the amino acid sequence from amino acid 62 to amino acid 71 of SEQ ID NO:2.
• such polynucleotide comprises the nucieotide sequence of SEQ ID NO:3 from nucieotide 85 to nucieotide 519; the nucieotide sequence of SEQ ID NO:3 from nucieotide 124 to nucieotide 519; the nucieotide sequence of the full-length protein coding sequence of clone huL105_7 deposited under accession number ATCC 98431; or the nucieotide sequence of a mature protein coding sequence of clone huL105_7 deposited under accession number ATCC 98431.
• the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone huL105_7 deposited under accession number ATCC 98431.
• the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 124 to amino acid 134.
• the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having chemokine activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:4, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having chemokine activity, the fragment comprising the amino acid sequence from amino acid 67 to amino acid 76 of SEQ ID NO:4.
• the polynucleotide is operably linked to an expression control sequence.
• the invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Processes are also provided for producing a protein, which comprise:
• the protein produced according to such methods is also provided by the present invention.
• inventions provide an isolated gene corresponding to the cDNA sequence of SEQ ID NO:l, and an isolated gene corresponding to the cDNA sequence of SEQ ID NO:3. Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein.
• compositions comprising a protein having chemokine activity are also disclosed.
• the protein comprises an amino acid sequence selected from the group consisting of:
• the present invention provides a protein compnsing a fragment of the amino acid sequence of SEQ ID NO:2 having chemokine activity, the fragment preferably compnsing eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO.2, or a protein comp ⁇ sing a fragment of the amino acid sequence of SEQ ID N0 2 having chemokine activity, the fragment comp ⁇ sing the amino acid sequence from amino acid 62 to amino acid 71 of SEQ ID NO.2
• such protein comp ⁇ ses the amino acid sequence of SEQ ID NO.4, the am o acid sequence of SEQ ID NO: 4 from amino acid 124 to ammo acid 134, or the ammo acid sequence of SEQ ID NO:4 from am o acid 14 to amino acid 145.
• the present invention provides a protein comp ⁇ smg a fragment of the amino acid sequence of SEQ ID NO:4 having chemokine activity, the fragment preferably comp ⁇ sing eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:4, or a protein comp ⁇ sing a fragment of the ammo acid sequence of SEQ ID NO:4having chemokine activity, the fragment comprising the amino acid sequence from amino acid 67 to amino acid 76 of SEQ ID NO:4.
• compositions may further comprise a pharmaceutically acceptable carrier.
• compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
• Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
• the sequence for the human LI 05 was found in the Genbank EST database by searching using the murine L105 sequence (polynucleotide, SEQ ID NO:5; protein, SEQ ID NO:6).
• the search revealed two possible related human ESTs, AA027314 (SEQ ID NO:7) and W17274 (SEQ ID NO:8), which partially overlapped.
• the coding sequence of huL105_3 includes nucleotides 80-481, which encode a 134 amino acid full-length protein.
• the mature protein begins at amino acid 14 (SEQ ID NO:2).
• the polynucleotide and amino acid sequences of huL105_7 is reported as SEQ ID NO:3 and SEQ ID NO:4, respectively.
• the coding sequence of huL105_7 includes nucleotides 85-519, which encode a 145 amino acid full-length protein.
• the mature protein begins at amino acid 14 (SEQ ID NO:4).
• huL105_3 and huL105_7 contain an insertion relative to huL105_3.
• the inserted region is found at nucleotides 454-486 of SEQ ID NO:3. This region encodes additional amino acids 124-134 in SEQ ID NO:4.
• huL105_3 and huL105_7 cDNAs were deposited on May 9, 1997 under the Budapest Treaty with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) and were assigned accession numbers ATCC 98429 and ATCC 98431, respectively.
• Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al, Bio/Technology J O, 773-778 (1992) and in R.S. McDowell, et al., J.
• fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.
• fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
• linker For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule.
• Other immunoglobulin isotypes may also be used to generate such fusions.
• a protein - IgM fusion would generate a decavalent form of the protein of the invention.
• the present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucieotide sequence of each disclosed clone.
• the mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
• the sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
• the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
• “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
• Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
• An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated. Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci.
• Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
• organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark etaL, 1994, Proc. Natl. Acad. Sci.
• Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
• Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention.
• a "species homologue” is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or
• polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide
• protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucieotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
• species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatto, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O
• allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides.
• allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by
• Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
• the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
• the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
• the hyb ⁇ d length is that anticipated for the hyb ⁇ dized reg ⁇ on(s) of the hyb ⁇ dizing polynucleotides
• the hyb ⁇ d length is assumed to be that of the hyb ⁇ dizing polynucleotide.
• the hyb ⁇ d length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions ot optimal sequence complementanty
• SSPE (lxSSPE is 0.15M NaCl, lOmM NaH 2 P0 4 , and 1.25mM EDTA, pH 7 4) can be substituted for SSC (lxSSC is 0.15M NaCl and 15mM sodium citrate) in the hybndization and wash buffers; washes are performed for 15 minutes after hybndization is complete.
• each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. .19, 4485-4490 (1991), in order to produce the protein recombinantly.
• an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. .19, 4485-4490 (1991)
• Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185. 537-566 (1990).
• operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
• Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL- 60, U937, HaK or Jurkat cells.
• monkey COS cells Chinese Hamster Ovary (CHO) cells
• human kidney 293 cells human epidermal A431 cells
• human Colo205 cells human Colo205 cells
• CV-1 cells other transformed primate cell lines
• normal diploid cells cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL- 60, U937, HaK or Jurkat cells.
• yeast eukaryotes
• prokaryotes such as bacteria.
• yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
• Potentially suitable bacterial strains include Escherichia coli. Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
• the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
• suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
• Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference.
• an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
• the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
• the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
• the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
• affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®
• hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether
• immunoaffinity chromatography immunoaffinity chromatography
• the protein of the invention may also be expressed in a form which will facilitate purification.
• it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
• MBP maltose binding protein
• GST glutathione-S-transferase
• TRX thioredoxin
• Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
• the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
• One such epitope (“Flag") is commercially available from Kod
• RP- HPLC reverse-phase high performance liquid chromatography
• hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
• Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
• the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.”
• the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucieotide sequence encoding the protein.
• the protein may also be produced by known conventional chemical synthesis.
• the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
• the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
• modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
• Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
• one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
• Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584).
• such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
• a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, lymphocytes (such as, for example, thymocytes, monocytes, neutrophils and T-cells), mast cells, eosinophils and/or endothelial cells.
• lymphocytes such as, for example, thymocytes, monocytes, neutrophils and T-cells
• mast cells eosinophils and/or endothelial cells.
• Murine LI 05 protein has been shown to be chemotactic for thymocytes, kidney mesangial cells and lymphocytes localized in lymph nodes. Human LI 05 protein shares these activities, as well as possessing other chemotactic and chemokinetic activities.
• Chemotactic and chemokinetic proteins can be used to mobilized or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
• the chemokinetic proteins of the present invention can also be used to block chemotaxis (e.g., by flooding the blood with the proteins and inducing "chemotactic paralysis" (i.e., there is no gradient for cells to move along, so no movement occurs)), such as to block inflammation.
• a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
• the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
• a protein has "chemokine activity” if it either (1) displays chemoattractant or chemotactic activity in a chemoattraction or chemotaxis assay (preferably an assay in which the corresponding species mature protein is active), or (2) displays biological activity in a factor-dependent cell proliferation assay (preferably an assay in which the corresponding species mature protein is active), or (3) displays activity in the induction of lymphokine production or effector function in an immune cell functional assay.
• effector function include, without limitation, tumoricidal activity, granule release, adhesion molecule expression, and the like.
• Chemoattractant or chemotactic activity can also be measured in vivo by injecting protein at a particular site and performing a histological examination of the cell types that migrate to the site of injection.
• the activity of a protein of the invention may, among other means, be measured by the following methods:
• Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
• Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Intersciece (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
• a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art.
• pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
• the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, JL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
• the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compliment its activity or use in treatment.
• protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
• a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
• pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
• the pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens.
• the protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes.
• B lymphocytes will respond to antigen through their surface immunoglobulin receptor.
• T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins.
• MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
• the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
• antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical compositon of the invention.
• the pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
• Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No.4,235,871 ; U.S. Patent No.
• the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
• a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
• the term refers to that ingredient alone.
• the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
• a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
• Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
• protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially.
• cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors are administered sequentially.
• Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous administration to the patient is preferred.
• protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
• the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
• the tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
• a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
• the liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
• the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
• protein of the present invention When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
• a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
• the pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
• the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
• compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about 0. l ⁇ g to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
• the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each apphcation of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
• Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
• the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).
• KLH keyhole limpet hemocyanin
• Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
• Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
• neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
• Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
• Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• FEATURE :
• MOLECULE TYPE protein
• SEQUENCE DESCRIPTION SEQ ID NO : 2 :
• CTGCTTTAAC CACCCCATCT GCATTCCCAG CTCTACCCTG CATGGCTGAG CTGCCCACAG 720

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• 1998
  • 1998-06-10 EP EP98932728A patent/EP0988319A1/de not_active Withdrawn
  • 1998-06-10 JP JP50308499A patent/JP2002504823A/ja active Pending
  • 1998-06-10 AU AU82545/98A patent/AU8254598A/en not_active Abandoned
  • 1998-06-10 CA CA002295982A patent/CA2295982A1/en not_active Abandoned
  • 1998-06-10 WO PCT/US1998/011908 patent/WO1998056818A1/en not_active Application Discontinuation

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